Interest in stem cell biology and its association with tissue engineering and regenerative medicine has grown rapidly over the past decade. Autologous, adult and cord-derived stem cells were the first to be implanted into patients for numerous purposes including haematological, musculoskeletal, and immunological disorders [1]. More recently, hESCs have also been approved for clinical trials in both macular degeneration and spinal cord injury, though we can anticipate that this list will grow over the coming years [2, 3]. Particular attention has been drawn towards hESC due to their pluripotentiality, immortality, and immunologically privileged nature [4]. These characteristics endow great potential as an off-the-shelf, allogeneic, scalable solution to numerous conditions, for which, adult and cord-derived stem cells are not applicable, due to limited differentiation and/or proliferative capacities [5].
Current methodology for expansion of hESCs is largely reliant on either the mitotically-inactivated feeder cell method (using direct co-culture with embryonic or adult fibroblasts), or the feeder-free method, which utilises feeder cell, pre-conditioned media and a biological substrate, such as Matrigel™ [4, 6]. Unlike with MSC culture, the presence of a feeder cell layer, Matrigel™ or other extracellular matrix component scaffold is considered essential for the culture of ESCs (see Ramirez et al., 2011), with “naked” or uncoated plastic being reportedly unable to support undifferentiated culture of hESCs (Xu et al Nature Biotechnology 2001), whereas mESC culture requires only a thin layer of collagen for self-renewal.
Fok and Zandstra (2005) described stirred-suspension culture systems for the propagation of undifferentiated mouse embryonic stem cells (mESCs). The stirred-suspension culture systems comprised microcarrier and aggregate cultures. Mouse embryonic stem cells cultured on glass microcarriers had population doubling times comparable to tissue-culture flask controls. Upon removal of leukemia inhibitory factor, the mESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. However, LIF does not have this effect on hESC culture (Smith et al, Nature (1988) 336: 688-690). Suspension cultures of mouse ESCs are also described in King and Miller (2005). However, King and Miller (2005) state that “expansion of undifferentiated human ESCs (hESCs) is more difficult than for mESCs and has not yet been reported in stirred cultures”.
Matrigel™ itself is a loosely defined substrate, which consists of extracellular matrix proteins from Engelbreth-Holm-Swarm tumours [6]. The inherent limitation of the Matrigel™-based feeder-free method is that it is unsuitable for incorporation into hESC-based clinical trials due to the risk of xenocontamination. In addition, Matrigel™ limits hESC expansion to a two dimensional (2D) environment with subsequent interventions required prior to transplantation. Hence, innovative and novel tissue engineering strategies are urgently required to provide the opportunity of incorporating hESCs with synthetic, biomimetic substrates (scaffolds), with the potential to act as three dimensional (3D) carriers to facilitate ready transplantation into in vivo target sites. Other extracellular matrix components used as a support for stem cell proliferation include laminin, vitronectin, fibronectin, hyaluronan and collagen, or mixtures of these components (see, for example, Ludwig et al., (2006) Nature Biotech 24(2): 185-87), Ramirez et al, 2011). Such mixtures are often only loosely defined in terms of their composition.
Cells are sensitive to nano-scale topography [7]. A common method used for fabricating nano-scale tissue engineering scaffolds is electrospinning. Electrospinning offers advantages over other nanofiber fabrication techniques such as phase separation, self assembly, and template synthesis, as it is fast, efficient, versatile and economical [8, 9]. Electrospinning provides the opportunity to produce nanofibrous scaffolds, of tailored dimensions, that are able to mimic the nano-architecture of native extracellular matrix [10]. Nanofibers also provide a high surface area to volume ratio and high surface roughness resulting in an effective environment for cell adhesion due to increased focal adhesion contact between the cells and the surrounding fibers [11-13]. This is an indication of effective interaction between the cell and the surrounding artificial ECM, which results in the potential transmission of guidance cues to the cells. Electrospun fiber meshes generally have poor mechanical strength, but are highly flexible, which can result in an environment where cells produce fewer stress fibers [14]. An involvement of nanotopographical features in the maintenance of undifferentiated ESCs has been previously proposed [15].
Electrospinning has proven to be of great interest in the field of tissue engineering [16, 17]. It permits fabrication of fibers in both random and aligned conformations, which can be applied to various surfaces and provides the opportunity to use natural or synthetic polymers [18]. Modulation of voltage, electrode-collector distance and polymer concentration allows control of fiber diameter, morphology and porosity of a scaffold/substrate [19].
Previous reports have detailed the biocompatibility of electrospun nanofiber scaffolds to support the attachment, proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (hMSC), cord blood-derived somatic stem cells, neural stem cells, and haematopoietic stem cells [13, 20-27]. Many of these researchers used synthetic polymers such as poly-ε-caprolactone (PCL), poly-L-lactide acid (PLLA), poly(lactic-co-glycolic acid) (PLGA), as they are FDA approved and their bulk degradation properties are well characterised [19]. hMSC was reported to form both osteogenic and chondrogenic lineages on randomly arranged PCL nanofiber scaffolds [20, 28]. In addition, neural stem cells were capable of differentiating into functional nerve cells with increased neurite outgrowth and reduced branching on PCL and collagen/PCL aligned nanofiber scaffolds [20, 23, 28]. Cord blood-derived somatic stem cells, on the other hand, were differentiated towards hepatocyte-like cells on random PCL nanofibers. Albumin, glycogen storage, and α-fetoprotein were all detected after six weeks of differentiation towards a hepatic lineage and RT-PCR analysis showed the presence of endodermal, hepatic genes [25]. These observations were recently expanded; aligned and random nanofiber scaffolds, fabricated using PCL, PLGA and poly-L/D-lactide acid (PLDLA), were demonstrated to being suitable for the isolation and expansion of hMSC directly from bone marrow aspirate while maintaining their multipotent state [27]. However, different types of stem cell may require different cultivation conditions, even if isolated from the same tissue (see Ulloa-Montoya et al., 2005), and so methods for the culture of MSCs are not directly transferrable to ESC or other types of stem cell. Currently, the literature regarding culture and differentiation of hESCs on electrospun fibers is limited. However, murine ESC differentiation into mature neural cells was achieved using retinoic acid, via an embryoid body (EB) stage, when cultured on electrospun, oriented PCL nanofiber scaffolds [29]. Immunostaining was performed to characterise the mature neural cells lineages, including neurons (Tuj-1), oligodendrocytes (O4) and astrocytes (glia fibrillar acidic protein; GFAP). Results showed that culture of EBs on nanofibers (both random and aligned) significantly enhanced the differentiation towards neural lineages. Culture of EBs on oriented nanofibers also significantly enhanced neurite outgrowth when compared to EBs cultured on random nanofibers [29]. Electrospun polyurethane porous nanofibers are described as supporting the proliferation and neuronal differentiation of hESC in previous studies [30]. In this instance, hESC were differentiated towards a neural lineage, as characterised by immunohistochemistry for MAP2, β-tubulin III, and tyrosine hydroxylase (TH). Lineage specificity was demonstrated by the relative absence of GFAP (astrocytes), and a negative immunoreaction for hESC markers Oct-4, Sox2 and Nestin indicated the absence of undifferentiated hESCs. Culture of hESC on electrospun nanofiber substrates fabricated from PCL/collagen and PCL/gelatin has also been described. However, hESC/mouse embryonic fibroblast (MEF) co-culture was the only method by which hESCs could be expanded in a pluripotent state [31].
There thus exists a need in the art to expand pluripotent stem cells such as hESC, in which pluripotency is maintained and which is free of xenocontaminants.